1. Field of the Invention
The present invention relates to magnetic micro-particles, a method and an apparatus for collecting specimens using an antigen-antibody reaction, a method and a device for preparing specimens, which are suitable for use in immunonological diagnosis, biotechnology such as cell engineering, as well as virology and immunology.
2. Prior Art
Development of immunoassay methods utilizing an antigen-antibody reaction is now being made on a global scale as an early detection method for new virus-based diseases such as acquired immune deficiency syndromes (AIDS) and adult T-cell leukemia as well as various cancers. Tests now practiced are antibody tests designed to detect antibodies produced by an immune reaction in humans after infection with immunogens and therefore they are disadvantageous in that even when infection occurred, presence of an infection is not judged during the latent period before an antibody is produced. For this reason, there is a keen demand for developing a test method which permits direct detection of viruses and allows early diagnosis.
As for the direct detection method for detecting viruses, a blood agglutination method has been known. This method, however, is poor in detection sensitivity, and it was necessary to cultivate and multiply viruses to a population of at least 10,000,000 individuals/ml, which operation is troublesome and time consuming. Furthermore, it was necessary in this method to find sensitive host cells suitable for cultivating viruses since host cells are different for different viruses. In addition, a search or screening of sensitive cells was difficult since cultivation of viruses infectious to humans using animal cells involves a technological bottleneck.
In the case of viral hepatitis, for example, there occurs very often accidents that medical workers are infected with virus from patients since the disease is notoriously infectious, causing a social problem. As for vital hepatitis, type A hepatitis virus and type B hepatitis virus are known as pathogens and vaccines have been prepared. However, no pathogenic virus has been found yet for non-A and non-B vital hepatitis despite the fact that the presence of such virus was supposed well before, and therefore, no accurate therapy has been established yet.
Morphological observation of virus for confirming its presence is achieved only by electron microscopic observation. The morphological observation makes it possible to judge which group the virus in question belongs to, and when a new virus is found, it is indispensable to finally confirm the virus under an electron miloscope. In this ease, however, it is difficult to make observation unless rims is present in a high concentration since virus is in the order of 20 to 200 nm in size and since a narrow region in the order of .mu.m is to be observed. Accordingly, it has heretofore been practically impossible to detect such a minute amount of virus that only about 10 individuals/ml are present in a solution. The same is true for diagnosis in the level of cells such as cancer cells.
In the case of cancers, initially only a single cell is cancerated and in a long period time the subject falls ill. Diagnosis therefor presently employed involving use of an endoscope, CT scanner, pathological tests, and the like is carried out mostly with the eyes of a doctor or physician, resulting in that at the moment when a subject was diagnosed to suffer cancer, that which is difficult to diagnose with one's eyes has already completed metabasis or metastasis, and a physical operation does seldom prevent palindromia. If diagnosis can be performed before palindromia takes place at a level of a cell, palindromia can be avoided and it will be possible to cure cancers by immunological therapy without operations. With view to this, immunological diagnosis for cancers such as one using a tumor marker is now under research and development although little is put in practice an early diagnosis method.
Biotechnology such as genetic engineering, cell engineering or a like has paid attention as an approach for finding a breakthrough to the difficult situation encountered in the medical field and bringing a technological innovation thereto. There is a possibility that with the use of biotechnological techniques put in practice, e.g., cell level immunological diagnosis techniques utilizing an antigen-antibody reaction, and gene level immunological diagnosis technique utilizing hybridization of DNA (formation of double strands) that early diagnosis of various diseases can be performed. For this reason, development of early diagnosis techniques is promoted on a global scale.
Upon carrying out research at a level of a virus, cancer cell or gene freely utilizing such biotechnological techniques, techniques are indispensable which enable detection and collection of specimens such as viruses, cancer cells, etc. which are the subject of the research.
In order to detect specimens with a high detection sensitivity, the present inventors previously studied laser magnetic immunoassay methods as described in WO/88/02118 (PCT/JP87/00694 corresponding to Japanese Patent Application Nos. 61-224567, (51-252427, (51-254164, (52-22062, (52-22063, 62-152791, 62-152792, and 62-184902), Japanese Patent , Application (Kokai) No. 1-107151 corresponding to Japanese Patent Application No. 62-264319, and Japanese Patent Application No. 62-267481. The methods are characterized by using a laser beam in detecting the presence of absence of an antigen-antibody reaction, using magnetic micro-particles as a labeling material and radiating a laser beam to a concentrated specimen and detecting reflected light or a like outgoing light from the specimen, and they permit ultramicro detection of specimens in the order of picograms. Particularly, the technology described in Japanese Patent Application No. 62-184902 applies a magnetic field to a solution containing a magnetic-labeled specimen to guide and concentrate the specimen to a laser beam radiation area on the surface of the solution using a magnet The guidance with a magnet causes minute protrusion on the surface of the solution and interference fringes generated in reflected light, the occurrence of which depends on the degree or height of the protrusion, are detected. This gives detection of the specimen. This method permits de:cotton of virus in a population in the order of 10 individuals/ml in contract to the conventional EIA in which detection is only possible when virus is present in a population in the order of 10,000,000 individuals/ml.
Based on the above-described assay method, the present inventors have labeled antigens or antibodies with magnetic micro-particles and carried out direct detection of viruses for the first time. It is now being confirmed that the laser magnetic immunoassay method has a detection sensitivity higher than RIA referred to hereinbelow which has been said to be most sensitive. For example, when performing virus detection experiments using inactivated type A and type B influenza viruses as a model for virus, detection of viruses in a population in the order of one individual/ml was successful as the present inventors reported in 35th General Meeting of Japan Virus Association (November of 1987, Lecture No. 4011, "Virus detection experiment using a newly developed immunoassay apparatus").
However, techniques for efficiently collecting specimens such as virus, cancer cells or lymphocytes have remained on the way of development and have not been put in practice yet.
On the other hand, examples of conventional micro-immunoassay methods that have been put in practice include radioimmunoassay (RIA), enzyme-immunoassay (EIA), fluorescence-immunoassay (HA), etc. These methods use antigens or antibodies which are labeled with an isotope, an enzyme or a fluorescent substance in order to detect the presence or absence of corresponding antibodies or antigens, respectively, that react specifically therewith.
RIA is a method in which the radioactivity of an isotope used as a label is measured to quantitatively determined the amount of the specimen which has contributed to the antigen-antibody reaction, and at present it is only RIA that permits ultramicro measurement at a detection sensitivity in the order of picograms. However, it has many restrictions in its practice because it uses radioactive substances as a label substance and needs special installment and because decrease of effect of labeling due to half-life and other factors must be taken into consideration. Furthermore, considering the present social environment in which disposal of radioactive wastes is a big social problem, practice of RIA is naturally restricted.
On the other hand, methods using enzymes or fluorescent substances as a label are methods in which the amount of the specimen which has contributed to the antigen-antibody reaction is measured by determining coloring or luminescence, and therefore do not have restrictions that arc imposed on RIA. However, the detection limit of these methods is in the order of nanograms.
As described above, among the conventional immunoassay methods, RIA, which has a high detection sensitivity, has many restrictions in practicing it due to use of radioactive substances, and on the other hand, EIA, FIA and the like methods, which are easy to practice, have a low detection sensitivity and they are applied mainly to tests for antibodies. Since tests for antibodies are designed to detect antibodies formed by immune reactions in humus, it is impossible in principle to directly detect viruses in blood.
In the labeling methods such as RIA, EIA and FIA described above, they are performed by adding to a specimen an excess amount of a labeling reagent such as a radioactive isotope, an enzymes or a fluorescent dye, respectively, and allowing an antigenantibody reaction between the specimen and the labeling reagent to occur, followed by removing unreacted labeling reagent by washing. Therefore, the more the amount of the specimen decreases the more excess the amount of the labeling reagent than the specimen, which causes a serious problem that the labeling reagent that remained unremoved after washing cause non-specific reactions.
Furthermore, application of laser magnetic immunoassay to electron microscopic observation of viruses, cancer cells, lymphocyte and the like specimens needs improvement in technology for the preparation of specimens in high efficiency.
Based on the results of detection for viruses using the laser magnetic immunoassay, the present inventors have presented lectures entitled "Detection of EB virus infected cells by magnetic Immunoassay", Lecture No. M-26, and "A new method for detection of virus antigens", Lecture No. M-31, in an international symposium on basis of chemotherapy of viruses and its clinical application held on Jun. 20, 1989 at Helsinki University in Finland.
The present inventors proposed to name the new method "MIA (magnet immunoassay) method". In order to take advantage of MIA method, it is important to develop a method for preparing specimens in which a very small amount of a specimen can surely be labeled magnetically.
Wolfgang Mueller Ruffoltz et al., Japanese Paint Application (Kokai) No. 61-293562, "Separator for magnetically removing magnetizable particles", disclose a separator using an electromagnet. The separator, which is designed to fractionate biological materials with resort to magnetic microspheres to remove cells, antigens, antibodies, enzymes, etc., is constructed such that Teflon piping called a separation unit: and having a length of 1 to 200 cm and a diameter 0.1 to 6 mm is wound so as to form a circle is fitted in an electromagnet, and is unsuitable if applied to preparation of specimens to be measured by the laser magnetic immunoassay method because a very large amount of magnetic-labeled antibody is to be used, thus failing to efficiently recovering specimens bound to the magnetic-labeled antibody.
A further problem of the separator is that if the length of the Teflon piping was reduced most of the specimen would pass through the piping without reacting with the magnetic-labeled antibody since the magnetic-labeled antibody is held only on the wall of the Teflon piping although the amount of the magnetic-labeled antibody to be used would be reduced.
A disadvantage common to the conventional met:hods is the fact that the smaller the size of ultramicro-particles of a magnetic substance the more difficult it is to surely hold the magnetic-labeled antibody in an electromagnet. That is, when ultramicro specimen such as a virus is labeled magnetically, it is preferred that the magnetic-labeled antibody is smaller than the virus. However it is known that the magnetic micro-particles composed of a ferromagnetic substance become non-ferromagnetic and converted to a superparamagnetic substance which does not respond to a magnet when their particle size becomes smaller. For example, magnetite is converted to superparamagnetic when the particle size is not larger than 10 nm, and when magnetic-labeled antibody is prepared using magnetite with a particle size of not larger than about 10 nm, it takes an hour or more according to the conventional methods to gather the magnetic-labeled antibody using a magnet.
Therefore, further improvement is desired.